1. Field of the Invention
The present invention relates to an autoradiographic process.
2. Description of the Prior Arts
There has been heretofore known a radiographic process termed "autoradiography" or "radioautography" comprising steps of: introducing a radioactively labeled substance into an organism; placing the organism or a part of tissue of the organism (that is, a sample or specimen) and a radiographic film such as a high-speed type X-ray film together in layers for a certain period of time to expose said film thereto; and obtaining the locational information on the radioactively labeled substance in said sample from the resolved pattern of the film. The autoradiography has been utilized, for example, to investigate the pathway and state of metabolism, absorption and excretion of the substance introduced in the organism in detail. Such autoradiography is described, for instance, in the following literature: Method in Biochemical Experiment, Volume 6, Method in Tracer Experiment I, 271-289, "8. Autoradiography" by Toru Sueyoshi & Akiyo Shigematsu (Tokyo Kagaku Dozin Ltd., 1977).
The autoradiography has been also utilized to obtain locational information on the radioactively labeled tissue of an organism and/or the radioactively labeled substances originating from an organism, which present on a medium. For instance, there is known an autoradiography comprising steps of: labeling organism-originating biopolymers such as proteins or nucleic acids with a radioactive element; resolving a mixture of the radioactively labeled biopolymers, derivatives thereof, cleavage products thereof, or synthetic products thereof on a support medium through a resolving process such as gel electrophoresis; placing the gel support and a high-speed X-ray film together in layers for a certain period of time to expose said film to the gel support, developing said film, obtaining the locational information on the radioactively labeled substances from the developed film, and then performing the identification of the polymeric substances, determination of molecular weight of the polymeric substances and isolation of the polymeric substances based on the obtained locational information.
Recently, the autoradiography has been effectively used especially for determining the base sequence of a nucleic acid such as DNA. Therefore, the autoradiography is thought to be a very usefull means in the field of structural determination of polymeric substances originating from organisms.
Nevertheless, such useful autoradiography is not free from several drawbacks in the practical use.
As described above, in the conventional autoradiography, a support medium containing radioactively labeled substances is brought into contact in the form of layers with a radiographic film such as a high-speed X-ray film for a given time so that the film is exposed to the radiation and then a visible image indicating the positions of the radioactive substances is obtained.
The primary drawback resides in that the exposure operation should be carried out at a low temperature (e.g., 0.degree. C. to -80.degree. C.), especially when the radiographic film is combined with a radiation intensifying screen, for a long period of time (e.g., several tens hours to several days). This is because intense radioactivity is not imparted to the substances to be labeled, a latent image in silver salt of the film formed by exposure to a radiation or light emission tends to fade at a relatively high temperature such as room temperature and to be undevelopable, and the silver salt is easily fogged chemically through migration of deleterious ingredients from the support medium carrying the sample thereto.
The second drawback resides in that the exposure operation ought to be done in a dry state to prevent the radiographic film from wetting and being chemically fogged which decrease the quality of an image.
When the image obtained by the autoradiography is fogged as described above, the accuracy of locational information on the radioactively labeled substances lowers. For these reasons, the procedure of the conventional autoradiography is complicated as a whole.
The third drawback resides in that the radiographic film is readily influenced by physical irritation and produces fogging under application of physical pressure caused by the contact of the film with the hands of operators or the instrument in the exposure operation. In order to avoid the occurrence of physical fogging on the radiographic film, high skill and caution must be taken in the handling of the film. In addition, the exposure over a long period of time causes natural radioactivities incorporated in the support medium to take part in the exposure of the radiographic film. Thus, the accuracy of the locational information on the labeled substances lowers. In order to eliminate such interference, parallel experiments using control samples are generally performed to find out proper exposure time, but such more experiments make the procedure more complicated.
Further, in the conventional autoradiography it is necessary to detect the location of the radioactively labeled substances on the visualized autoradiograph by eye observation to obtain the desired information and a long time is taken for such visual detection.
To solve the above-described problems attached to the conventional autoradiography, an autoradiographic process using a stimulable phosphor sheet comprising a stimulable phosphor as a radiosensitive material in place of the radiographic film and kits employed therefor are described in Japanese patent application No. 57(1982)-193418, No. 57(1982)-193419 and No. 58(1983)-30604 (corresponding to U.S. patent application Ser. No. 549,417 or European patent application No. 83110984.8). One of the kits is a separation type which comprises a stimulable phosphor sheet and a support medium for resolution, and the other one is an integrated type which comprises a stimulable phosphor sheet and a support medium provided thereon.
The stimulable phosphor sheet is also called a radiation image storage panel, disclosed in, for example, U.S. Pat. No. 4,239,968 and thus its general constitution is already known.
The stimulable phosphor sheet comprises a stimulable phosphor, in which said phosphor is capable of absorbing radiation energy having passed through an object or radiated from an object; and releasing the radiation energy stored therein as stimulated emission when said sheet is excited with an electromagnetic wave (stimulating rays) such as visible or infrared rays. The stimulated emission is photoelectrically detected to obtain electric signals, which is then reproduced as a visible image on a display device such as CRT or on a recording medium such as a photographic film, or represented locational information in the form of symbols and/or numerals.
According to the autoradiographic process using the stimulable phosphor sheet, not only the exposure time is greatly shortened but also the accuracy of the locational information on the radioactively labeled substances is not lowered even when the exposure is carried out at an ambient temperature or a temperature therearound. The exposure operation previously taking many hours under chilled condition, is made easy and hence, the autoradiographic procedure can be greatly simplified.
Further, the employment of the stimulable phosphor sheet in the autoradiography as a radiosensitive material substantially prevents either the chemical fog or the physical fog, both of which are the unavoidable problems in the use of a conventional radiographic film. This provides an advantageous feature in the improvement of the accuracy of the locational information and workability of the autoradiography. It is also possible to easily reduce or eliminate such disadvantageous effect on the accuracy that is caused by the natural radioactivity or the radioactivity of impurities contained in the support medium, by applying a certain electric processing to the locational information stored in the stimulable phosphor sheet.
Furthermore, the visualization is not always required to obtain the locational information on the radioactively labeled substances which are stored and recorded on the stimulable phosphor sheet, that is, the information can be obtained in the desired forms such as a visible image, symbols and/or numerical values and combinations thereof by scanning the phosphor sheet with stimulating rays such as a laser to read out the locational information. It is also possible to obtain the required information in various forms by further processing the obtained image information by use of an appropriate electric means. Namely, the information can be obtained as an alternative information by subjecting the electric signals or A/D converted digital signals having the image information to certain signal processing. For example, the electric signals or digital signals having the locational information on the labeled substances may be analyzed by means of a computer etc. to directly obtain a desired information on the organism.
The signal processing method for digital signals to obtain the locational information on the radioactively labeled substances in the form of signals or numerals, is described in Japanese patent application No. 58(1983)-1327 (corresponding to U.S. patent application Ser. No. 568,877 or European patent application No. 84100144.9), etc. This method comprises obtaining the locational information on the radioactively labeled substances (for instance, radioactively labeled DNA fragments) resolved one-dimensionally on a support medium as digital signals and then subjecting the digital signals to a signal processing, to obtain the locational information (for instance, DNA sequencing) in the desired form of symbols and/or numerals. In the specification there is also described a method for visualizing the resulting electric signals or digital signals in the form of an image by using a reproducing and recording means. Thus, obtaining the locational information on the labeled substances as a visible image makes possible to compare the information in the form of symbols or numerals with the visible image. Further, the obtained visible image can be compared with another visualized autoradiograph.
Until now, the conventional radiographic method has been almost predominantly utilized for autoradiography, so that it is requested to obtain the locational information on the radioactively labeled substances in the form of an image, which can be directly compared with another visible image obtained by the conventional method. Thus, it is desired to preserve the obtained locational information in the form of such an image.
The above-described method, however, requires an additional apparatus for the visualization of locational information on the radioactively labeled substances. Particularly, the complication of such an apparatus is inevitable as far as a visible image in a preservable form which can be easily compared with other visible images is necessarily obtained.